An emulsion is a heterogeneous system consisting of at least two immiscible liquids, one of which is dispersed in the other in the form of droplets. Such systems possess minimal stability..sup.1 The problem of stability of the emulsion has conventionally been addressed by the addition of additives such as emulsifiers and finely divided solids. Emulsions consist of continuous and discontinuous phases. The discontinuous phase is referred to variously as the dispersed or internal phase, whereas the phase in which the dispersion occurs is referred to as the continuous or external phase. The standard components of emulsions are an oily and an aqueous phase. When water is the continuous phase, the emulsion is referred to as oil-in-water (o/w), and when oil is the continuous phase, the emulsion is referred to as water-in-oil (w/o). Multi-phase emulsions of water-in-oil-in-water (w/o/w) have gained an importance recently.
Oil-in-water emulsions are the most frequently used emulsions. However, water-in-oil and multiple emulsions are desirable for many applications and would be more extensively used if the problems with instability could be overcome. The Encyclopedia of Emulsion Technology states "Multiple (or double) emulsions are used as depot systems and can be considered as a variation of W/O type. Their potential advantages in drug delivery can be counter-balanced by an increased complexity of the dosage form and the attendant problems of optimal formulation and acceptable stability.".sup.2 W/O is an abbreviation for water-in-oil. "Despite their early promise, the multiple-emulsion system has not been widely utilized. . . . Multiple emulsions produced from vegetable oils are particularly difficult to make if a high yield of multiple droplets and good stability are required."
Emulsions generally consist of three components. The oil phase, the aqueous phase, and an emulsifier. Each of these components and the method in which they are prepared and combined contributes to the type and stability of emulsion. Many attempts have been made to define rules which determine the type of emulsion produced..sup.3 In general, the liquid (oil or water) in which the surfactant is most soluble will be the continuous phase in the final emulsion. However, correlations between the properties of surfactant, oil and other materials, and emulsification are very empirical. That is, the rules apply to a limited spectrum of materials and there are frequent exceptions..sup.4
The mode of mixing the components is important in determining the properties of emulsions. There are three general means of emulsification: 1) Mechanical, 2) Phase inversion, and 3) Spontaneous emulsification..sup.5
Mechanical emulsification which uses shear force to break the emulsion components into small particles is the most commonly used. Phase inversion refers to the process of producing an emulsion of one type, water-in-oil, with components which are most stable with the opposite type, oil-in-water. The emulsion then spontaneously changes type. These emulsions can be critically dependent upon concentration of materials, salts, temperature and other factors. Spontaneous emulsification refers to the situation where an emulsion is formed with minimal agitation..sup.6 This implies a level of thermodynamic stability which is highly desirable, but seldom achieved.
Surface active agents are compounds which contain a hydrophilic and hydrophobic moiety in the same molecule. They preferentially localize at interfaces between oil and water where they reduce the surface-free energy. Within the spectrum of surface active agents, however, there is great diversity of chemical structure and function. The concept of hydrophile-lipophile balance was developed in an effort to predict the function of surface active agents for making various types of emulsions and for other activities. Hydrophilic-lipophilic balance is a semi-empiric measurement of the relative strengths of the hydrophilic and hydrophobic components..sup.7 It is related to the free energy associated with positioning the amphyphilic molecule at the oil-water interface. The hydrophile-lipophile balance values required for various applications are shown in the following table:
TABLE I ______________________________________ Hydrophilic-Lipophilic Balance Ranges and Applications.sup.8 Range Application ______________________________________ 3 to 6 water-in oil emulsifier 7 to 9 wetting agent 8 to 13 oil-in-water emulsifier 13 to 15 detergent 15 to 18 solubilizer ______________________________________
These values are widely quoted in the literature as guides to the selection of emulsifiers for particular purposes. They are designed for use with nonionic emulsifiers. Analogous systems have been developed for anionic or cationic emulsifiers, but they are less useful than those for nonionic emulsifiers. Hydrophilic-lipophilic balance numbers have been published for many nonionic surfactants..sup.9
In addition, Davies devised a method for calculating hydrophile-lipophile balance numbers for surfactants directly from their formula using empirically derived numbers..sup.10 Thus, a group number is assigned to various component groups in emulsifiers and the hydrophile-lipophile balance (HLB) is then calculated from the following relation:.sup.11 EQU HLB=7+.SIGMA. (hydrophilic group numbers)-.SIGMA. (lipophilic group numbers):
HLB numbers have proved valuable guides for selecting emulsifiers in that emulsifiers outside of the specified range will seldom be efficacious for a particular application. However, a correct HLB number does not guarantee performance indicating that factors other than HLB are also important and must be considered. It should be noted that no applications are listed for surfactants with HLBs of less than three. Such agents have been used to spread bath oil on the surface of water, as vaccine adjuvants and for a few other applications. However, they are seldom used as emulsifying agents.
Most water-in-oil emulsions use surfactants with an HLB in the range of 3 to 6 as described above..sup.12 Finely divided solids may also be used as emulsifying agents. It has been reported that the objective in a water-in-oil emulsion is to produce an interfacial film with rigidity and no charge..sup.13 The stability of an emulsion can frequently be increased by increasing the concentration of emulsifying agent, but this increases problems of toxicity for biologic applications and may be suboptimal for other applications as well.
Emulsion stability is frequently increased when two surfactants with moderate differences in HLB and other properties are mixed together. If the differences are too large, however, then the combination seldom works. Attempts have been made to predict the function of blends of surfactants by averaging their HLB values, but instances are well known where blends produce results which are far different from the average of the components..sup.14 Mixed surfactants may produce a synergistic effect in stabilizing emulsions. This is thought to be related to the formation of structured complexes similar to liquid crystals at the oil-water interface. Phospholipids have been mixed with Span 80 to stabilize water-in-vegetable oil emulsions..sup.5
Finally, heterogeneity of polyoxyethylene chain length frequently effects emulsion stability, especially in situations where the surfactant is marginally soluble. Preparations with increased heterogeneity of chain length tend to produce emulsions with greater stability. As can be seen from the foregoing discussion, it is difficult to predict the emulsifying properties of any combination of compounds based on the physical properties of the compounds.
Various oils differ markedly in the ease with which they can be emulsified and in the stability of the resulting emulsions. In general, mineral oil is easier to emulsify than vegetable oils..sup.16 Each oil has a required HLB number for production of a particular type of emulsion. This is the reason for the range of HLB numbers for production of each type of emulsion as shown in Table I. The more polar the oil phase, the more polar the surfactant must be to produce an optimum emulsion. Water-in-oil emulsions follow similar rules as oil-in-water emulsions, but the HLB values are lower. HLB values required to produce water-in-oil and oil-in-water emulsions with many commonly used oils have been published..sup.17
Numerous methods have been devised for producing water-in-oil emulsions. Most of these involved mixing the components in a machine which produces agitation or a strong shear force. It has been recommended that oil-soluble materials be placed in the oil phase and water-soluble materials in the water phase prior to combining the phases..sup.18 However, this results in a less than satisfactory emulsion preparation. An exception to this is that very fine oil-in-water emulsions can be produced by placing a water soluble surfactant in the oil phase..sup.19 The reverse has not been recommended for water-in-oil emulsions. Placing hydrophobic surfactants in tie aqueous phase of an emulsion generally leads to poor emulsification. Fine oil-in-water emulsions can be prepared from water-in-oil emulsions by phase inversion. Many surfactants change their properties and switch from promoting one type of emulsion to the other with change in temperature..sup.20 This phenomena can be used to produce emulsions under certain circumstances.
Stabilizers for Water-in-Oil Emulsions
Prior art water-in-oil emulsions are difficult to stabilize. This has seriously impeded their application in many situations where they would otherwise be highly desirable. Stabilization has been attempted by increasing the viscosity of one or more of the phases or interfaces. That can be accomplished by adding polymeric stabilizers which form gels in the bulk phases or other structures at interfaces. Such polymers include proteins, starches, gums, cellulosics, polyvinyl alcohols, polyacrylic acid and polyvinyl pyrrolidone..sup.21 These materials bind to the interface by covalent bonds or by electrostatic and hydrophobic interactions. They form an "interfacial complex" which is defined as an association of two or more surface active molecules at an interface in a relationship that does not exist in either of the bulk phases. Certain complexes localized at the interface of oil-in-water in emulsions can be effective in stabilizing the emulsions. Some low molecular weight materials have similarly been used as stabilizers. These include cholesterol, which may form complexes with certain nonionic emulsifying agents..sup.22 In addition, fatty acid salts of divalent cations such as aluminum stearate are very effective in stabilizing water-in-oil emulsions. Salts of the same fatty acids with monovalent cations such as sodium stearate are not effective.
Problems with Prior Art Water-in-Oil Emulsions
The major problems impeding the increased use of water-in-oil emulsions are difficulty in preparation, high viscosity, and poor stability. Vegetable oils, such as peanut oil and soybean oil, or animal oil, such as squalene or squalane, would be preferable to mineral oil for many applications. However, they are more difficult to emulsify and make less stable emulsions. Water-in-oil emulsions of vegetable oil require relatively high concentrations of a hydrophobic emulsifier such as Span 80 and a stabilizer. Aluminum or magnesium stearate have been used successfully as stabilizers. However, they add to the complexity and toxicity of emulsions. Another problem is that the solubility of nonionic surfactants and thus their ability to produce stable emulsions varies with temperature. This produces problems in storage of emulsions where the temperature may vary from below freezing to greater than 120.degree. F.
Many attempts to overcome these problems have involved polymerization of some component of the emulsion to produce increased rigidity. Polymers have been added to either the aqueous or oil phases which are then polymerized chemically or by radiation. Some surfactants can be polymerized at the oil-water interface with x-radiation. This is not useful for water-in-oil emulsions which require hydrophobic surfactants because these surfactants tend to be degraded by x-radiation..sup.23 Finally, heating to temperatures which denature proteins is required to melt or dissolve certain components of emulsions..sup.24 This precludes the use of such emulsions for many biologic activities where native, non-damaged proteins are required. Finally, very high concentrations of emulsifying agents may be needed. In one example, up to 82% of the oil phase of the emulsions was made up of the surfactant Pluronic.RTM. L121 (poloxamer 401)..sup.25 Other examples in the same patent required heating to high temperatures to produce water-in-oil emulsions with block copolymer surfactants.
What is needed is an improved method of producing water-in-oil emulsions which does not require high temperatures, organic solvents, x-radiation or chemical reactions to cause crosslinking to form the desired emulsion. The method should optimally produce water-in-oil emulsions by spontaneous emulsification. In addition, the emulsions need increased stability. They should be stable at a high range of temperatures. Preferably, the emulsions should have lower toxicity to be useful for biologic applications. To do this, they should contain fewer components and the components that are present should be less toxic. Stabilizers should be reduced. The concentration of water in the water-in-oil emulsion should be increased to a higher level, preferably over 80%. Most water-in-oil emulsions have used less than 50% water. Finally, the water-in-oil emulsion should provide a good starting material for production of water-in-oil-in-water multiple emulsions.
Multiple Emulsions
The water-in-oil-in-water multiple emulsion comprises three distinct phases..sup.26 The innermost phase is aqueous. It is encapsulated in an oil phase which is, in turn, enclosed within a second aqueous phase. Each dispersed oil globule in a water-in-oil-in-water emulsion forms a vesicular structure with single or multiple aqueous compartments separated from the aqueous suspending fluid by a layer of oil phase components. Such emulsions have most of the advantages of water-in-oil emulsions with much lower viscosity. They also have many similarities with liposomes but have large aqueous compartments and can be prepared without inorganic solvents. Multiple emulsions can be considered a relatively unstable metaphase between water-in-oil and oil-in-water emulsions. The oil layer which separates the two aqueous phases may become very thin which is independent of the amount of the oil phase component(s). The stability of these emulsions may be phenomenologically understood as being brought about by the durability of the oil layer..sup.27 Rigid oil layers or films are associated by increased stability.
Multiple emulsions can be useful in many technologies, particularly in the pharmaceutical and in separation science. Their potential biopharmaceutical applications are unique as a consequence of the dispersal of one aqueous phase inside droplets of another. These include potential as vaccine adjuvants, drug delivery systems, sorbent reservoirs in drug overdose treatments and for immobilization of enzymes. They can also be used for separation of hydrocarbons and in the removal of toxic materials from waste water. Multiple emulsions according to the present invention can be formulated as cosmetics and as household products such as wax polish. They have been used to immobilize insulin for depot injection and in foods. The main problem associated with multiple emulsions is their instability which has severely limited their usefulness in the many applications for which they have shown obvious promise..sup.28
Production of Multiple Emulsions
Multiple emulsions have been produced by several techniques which have advantages in different situations. However, none of them are optimal. One procedure involves the preparation of a water-in-oil emulsion which is then converted to a multiple emulsion by increasing the amount of water phase until the emulsifying capacity of the oil phase is exceeded. Alternatively, by changing the temperature past the inversion point, some emulsions will transform from water-in-oil to an oil-in-water producing a transient multiple emulsion phase.
Multiple emulsions having two different aqueous phases must be prepared in two steps. This is accomplished by producing a water-in-oil emulsion by any standard technique. This water-in-oil emulsion is then re-emulsified in the second aqueous phase which normally contains surfactants. Nonionic emulsifiers are usually preferable to ionic ones for the second step. Span 80 has been a successful emulsifier for the water-in-oil emulsion in the first step. "A large amount of Span 80 in liquid paraffin, however, is one of the necessary conditions for developing multiple emulsion type dispersions.".sup.29 Less than 20% Span 80 results in an oil-in-water emulsion (see Table II). The optimal for paraffin oil is approximately 30%. For emulsification of animal oil or vegetable oils, even larger amounts of Span 80, in the order of 50%, are required. The water-in-oil emulsifier is always added to the oil phase of the emulsion. The concentration of hydrophilic emulsifier in the outer aqueous phase is also critical in that the concentration must be very small in relation to the concentration of hydrophobic emulsifier in the oil phase. For example, concentration of Tween 80 in the aqueous phase cannot exceed 1%, while Span 80 in the oil phase must be in excess of 30% to produce stable emulsion..sup.30
TABLE II ______________________________________ Type of Multiple Emulsion % Span 80 Emulsion Index ______________________________________ 10 o/w 0 20 o/w 0 30 w/o/w 0.7 40 w/o/w .15 50 w/o/w .25 60 w/o/w .30 70 w/o/w .27 80 w/o/w .16 90 w/o/w .05 100 w/o/w 0 ______________________________________ Table II: Effect of Span 80 concentration in the oil phase on the formation of waterliquid paraffinwater emulsions prepared by the mechanical agitation; concentration of SDS in the aqueous phase was fixed at 0.15M in all instances. The multiple emulsion index is a measure of th formation of w/o/w..sup.31
A variety of stabilizing agents and regimens have been used in the prior art to increase the stability of multiple emulsions. Soy lecithin at a concentration of 8% allowed a reduction in the amount of Span 80 to 20% while maintaining stability of the emulsion. Like water-in-oil emulsions, the volume fraction of the oil phase is not critical. This is thought to be due to the fact that emulsion components are mobile and stability depends upon maintaining integrity of a lipid film as it thins..sup.32
Like water-in-oil emulsions, vegetable oils and animal oils are more difficult to use in production of multiple emulsions than mineral oil. For example, 60% Span 80 is required to produce a multiple emulsion with olive oil..sup.33 This can be reduced to 43% if 17% soy lecithin is added. In addition, pairs of surfactants can be used to stabilize multiple emulsions in much the same manner that they have been used for w/o emulsions.
Block Copolymer Surfactants in Prior Art Multiple Emulsions
Copolymer Pluronic.RTM. L101 (poloxamer 331) has been used as the hydrophobic emulsifier in combination with Pluronic.RTM. P123 (poloxamer 403) as the hydrophilic emulsifier to produce water-in-oil-in-water multiple emulsions..sup.34,35 The Pluronic.RTM. L101 at 5% in mineral oil was emulsified with saline containing 2% bovine serum albumin. Over time, the bovine serum albumin formed a complex with the Pluronic.RTM. L101 at the oil-water interface. This complex was thought to be important in maintaining the stability of the water-in-oil component of the emulsion. If the multiple emulsion is produced before this interfacial complex has time to form, the resulting water-in-oil-in-water emulsion is less stable. This emulsion was then re-emulsified in saline containing 0.4% Pluronic.RTM. P123. Several copolymer surfactants were evaluated in this study. Pluronic.RTM. L101 and P123 were found to be the most effective. Copolymers with hydrophobes larger than 4000 have not been evaluated.
Physical Stabilizers
Because of the inherent instability of multiple emulsions, several approaches to enhancing stability by producing physical rigidity have been tried..sup.36,37 One approach utilizes interaction between hydrophilic polymers in the inner aqueous phase with the surfactant. The BSA-L101 interaction described above is an example. Other examples used polyacrylic acid or polyelectrolytes with high molecular weights approaching three million. In addition, the surfactant on the interface has been crosslinked by x-radiation or polymerization of chemically reactive monomers..sup.38 This has been done to polymerize material in the inner aqueous phase, at the oil-water interface, in the oil phase, and in the outer aqueous phase. The oil droplets have been encapsulated with gelatin and other materials to provide physical barriers. While each of these methods has provided a measure of increased stability under certain conditions, the emulsions have seldom been sufficiently stable, nontoxic and functional to facilitate widespread use.
Problems with Multiple Water-in-Oil-in-Water Emulsions
As mentioned above, the primary problem in water-in-oil-in-water emulsions is stability. The inherent instability of multiple emulsions has precluded most commercial uses. However, there have been a few reports of attempts to improve stability. Procedures for increasing the stability of multiple emulsions have been rather empirical in that each manipulation tends to be highly specific for the particular emulsion under evaluation..sup.39 Nevertheless, some principals can be formulated. The most important problem appears to be the instability caused by the aqueous emulsifier in the outer layer which progressively solubilizes the hydrophobic emulsifier in the oil layer and destroys the internal emulsion..sup.40 The almost inevitable interaction of the surfactant used in the second emulsification step with the initial interfacial film demonstrates the inherent impracticality of employing free surfactant stabilizers in multiple emulsions. A more permanent interfacial membrane which does not permit diffusion of stabilizing components is preferable.
Assessment of Multiple Emulsions
Multiple emulsions have been classified as types A, B and C, depending upon the size and number of water droplets inside of the oil drops. Type A has a single water droplet, B a small number, and C a larger number of water drops within the oil drops..sup.41 According to this classification, the emulsions produced as Freund's complete adjuvant or the multiple emulsions of the present invention are much finer than even the C emulsions. It has been reported that multiple emulsions may be more stable than the original water-in-oil emulsions under certain conditions. In one example, soybean oil emulsified with glyceryl monooleate was stable at 80.degree. C..sup.42 It is reported that multiple emulsions made with Freund's adjuvant are more stable than the parent water-in-oil emulsion in storage at 4.degree. C..sup.43
The overriding problem limiting the usefulness of multiple emulsions is stability. Most publications reporting on stability demonstrate unacceptable levels even for the more stable preparations. For biologic products, the emulsions should have a shelf life comparable to that of the contained drug in the refrigerator or freezer. In addition, many of the components used to increase stability will also increase toxicity. Multiple emulsions have usually used high concentrations of hydrophobic surfactants and stabilizers which are inherently toxic. In addition, a low concentration of water, less than 50% in the internal aqueous phase, forces an increase in the amount of oil for the amount of inner aqueous phase to be delivered. This is important because the inner aqueous phase is the site of choice for most active ingredients. Finally, it would be highly desirable to use a vegetable oil such as peanut oil or an animal oil (squalene or squalane) in place of the non-metabolizable mineral oil. However, multiple emulsions with vegetable oil have been even more difficult to produce and maintain stable than those with mineral oil.